POS0098 IDENTIFICATION OF THE CENTRAL TOLERANCE CHECKPOINT FOR AUTOREACTIVE PROTEINASE 3+ B CELLS IN HUMAN BONE MARROW
Authors: A. Berti, M. Tomasi, I. Pesce, E. Lista, A. Guella, G. Paolazzi, R. Bortolotti, G. Grandi, S. Hillion, U. Specks, D. Cornec
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Autoreactive proteinase 3 (PR3+) B cells have recently been phenotypically and functionally characterized, and the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with ANCA-associated vasculitis (AAV) has been shown. This work aimed to investigate the central tolerance-checkpoint controlling immature PR3+ B cells in the bone marrow (BM), before their migration into the periphery as transitional B cells. <h3>Objectives</h3> We investigated the presence and the specific phenotypic features of PR3+ B cells in BM mononuclear cells (BMMC) of non-vasculitis controls (No-AAV), comparing them to paired peripheral blood mononuclear cells (PBMC) of No-AAV and PBMC of PR3-AAV patients, and the central tolerance-checkpoint for PR3+ B cells. <h3>Methods</h3> We used a customized flow-cytometry assay, using PR3 as ligand to target autoreactive PR3+ B cells (PR3+B cells). Adult PR3-ANCA positive AAV (PR3-AAV) patients with a clinical diagnosis of granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) were selected among consecutive subjects with AAV seen in our Rheumatology Unit. Subjects without vasculitis (No AAV) were selected among consecutive subjects undergoing bone marrow aspirate to exclude hematologic conditions of myeloid origin and eventually resulted healthy or in long-term complete remission during follow-up of myeloid neoplasms. PMBC from AAV patients and paired samples of BMMC and PBMC from No AAV were collected and analyzed. <h3>Results</h3> The proportion of PR3+ B cells within BMMC (median [IQR25-75%]; 1.98%[1.77-2.75]) was higher than within PBMC of No-AAV (0.9%[0.63-1.44], p&lt;0.01 by paired comparison) and similar to their proportion within PBMC of PR3-AAV patients (1.82%[1.66-3.21]; p&gt;0.05). When focusing on immature/transitional CD24++CD38++B cells only in No-AAV, we observed distinct phenotypes within BMMC versus PBMC (i.e. higher proportion of CD27-CD10+ and lower expression of CD21, IgD, IgM within BMMC versus PBMC), representing two separate developmental steps of B cell maturation. Within CD24++CD38++ B cells, BMMC contained the greatest proportion of PR3+ B cells as compared to PBMC (3.35%[1.99-4.92] versus 1.23%[0.62-1.55], p&lt;0.01). We observed a significant decline of the PR3+ fraction from T1-like/immature subset (IgD-IgM+; 2.80%[1.23-4.02]) to T2-like/early transitional subset (IgD+IgM+; 1.76%[0.96-2.68], p&lt;0.01) in BMMC, while no significant reduction was observed between the latter subset and the transitional compartment of PBMC (1.26%[0.62-1.56], p&gt;0.05). <h3>Conclusion</h3> To prevent PR3-related autoimmunity, autoreactive PR3+ B cells pass a stringent selection in the BM, and their removal by central tolerance-checkpoint activity occurs mainly between T1-like/immature to T2-like/early transitional B cells of BMMC. <h3>References</h3> [1]Cornec D. Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls. J of Autoimmunity, 2017. [2]Berti A. Circulating autoreactive proteinase 3+ B cells and tolerance checkpoints in ANCA-associated vasculitis. JCI Insight, 2021. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Alvise Berti Speakers bureau: GSK, Michele Tomasi: None declared, Isabella Pesce: None declared, Enrico Lista: None declared, Anna Guella: None declared, Giuseppe Paolazzi: None declared, Roberto Bortolotti: None declared, Guido Grandi: None declared, Sophie Hillion: None declared, Ulrich Specks: None declared, Divi Cornec: None declared.